The Isolation, Cultivation and Identification of Viruses

Viruses must be grown in living cells. They can't be grown
in culture media or on agar plates alone, they must have living cells to support
their replication.

The easiest viruses to grow are bacteriophages (because the
easiest cells to grow in the lab are bacteria).

Growing Bacteriophages In The Laboratory

Once viruses have replicated and been harvested the concentration
of viral particles (virions) in the viral stock solution must be determined.
One of the easiest ways to determine the concentration of a stock solution
of bacteriophages is to use the plaque method.

The plaque method:

Virus, bacteria, and agar mixed, plated and incubated.

After replication the virus lyses the bacteria, forming
plaques, or clear zones.

Each plaque is assumed to come from a single viral particle.

The titer (concentration of the stock solution) of the virus
is given in plaque forming units.

3. Cell culture is a lot cheaper and easier to work with (contamination
can be a problem however).

Primary cell lines have a short lifespan in culture –
a few generations before reaching senescence.

Diploid cell lines are derived from embryos and can grow
for up to 100 population doublings before senescence.

Continuous cell lines are derived from transformed cells
and grow indefinitely in culture.

Hela cells – 1st continuous cell line, derived
from Helen Lane (fictional name - actually named Henrietta Lacks), a cervical
cancer patient who died in 1951. This is the oldest continuous cell line
and was first used to culture and identify polio virus.

Transformed Cells in Culture

Viral growth can cause cytopathic effects in the cell culture.

Cytopathic effects can appear early or late in the course of
the viral infection.

Cytopathic effects may be cytocidal (cell death) or non-cytocidal.

Non-cytocidal effects include acidophilic or basophilic inclusion
bodies in the nucleus, cytoplasm, or both; cell fusion, and transformation.

Cytopathic
effects can be so characteristic of individual viruses that they can often
be used to identify viruses.

(a)
Uninfected cells in culture form a monolayer

(b)
Cells infected with vesicular stomatitis virus round up and pile up on top of
each other